Controlled Release of Multiple Growth Factors in 3D-Printed Scaffolds

1Poldervaart, M T; 2Wang, H; 2Leeuwenburgh, S C; 1Öner, F C; 1Dhert, W J A; +1Alblas, J 1Department of Orthopaedics, University Medical Center Utrecht, The Netherlands

2Department of Biomaterials, Radboud University Nijmegen Medical Center, The Netherlands j.alblas@umcutrecht.nl

INTRODUCTION Several regenerative strategies are currently under development as alternatives to bone grafts. Cell-free constructs, based on a scaffold loaded with multiple growth factors seem to be an attractive alternative. Different growth factors can be combined in a single spatially organized scaffold. This can be achieved by 3D fiber deposition (3DF), a technology to build hydrogel scaffolds in a reproducible and precise manner [1]. Candidates for cell recruitment and osteogenic differentiation are stromal cell-derived factor 1α (SDF-1α) and bone morphogenetic proteins (BMPs) respectively. The chemokine SDF-1α is able to recruit allogeneic MSCs to a tissue engineered construct [2] and plays a role in vascularization, a process necessary for bone formation, especially in clinically relevant sized scaffolds. Vascular endothelial growth factor (VEGF) has been shown to act synergistically with BMP-2 [3] to result in osteogenic differentiation. BMP-2 is currently used at supraphysiological doses in the clinic, which may explain several side effects, leading to controversy. The used dosage is much higher than the ED50 to compensate for the fast degradation of the protein. Gradual release of the growth factors at a specific location is probably more efficient, hereby lowering the dose and costs. To accomplish such a release system, microparticles based on gelatin as a non-cytotoxic, biodegradable natural product, are of interest. We aim for a prolonged and continuous delivery of BMP-2 combined with a faster release of VEGF and SDF1α using gelatin microparticles. The second aim is to print the microparticles in 3D porous hydrogel constructs in order to reach cell-free bone regeneration. METHODS Gelatin microparticles (GMPs) are produced by adding hydrated gelatin to refined olive oil, creating a water-in-oil emulsion. The solution is stirred, then rapidly cooled, washed with acetone and filtered. Particles were sieved and covalently cross-linked using 25 mM glutaraldehyde and subsequently washed with 100 mM glycine and water. After this the particles were freeze-dried. Growth factors were dissolved (range 10/125ng growth factor/mg GMPs) in PBS/BSA and then loaded onto the microspheres by diffusion. BMP-2 release (n=4) from GMPs was measured by loading 300 ng of 125I- labeled BMP-2 (InductOs, Medtronic) per sample of 5 mg GMPs. At each time point GMPs were centrifuged and supernatant was removed and replaced. In these samples 125I-BMP2 concentration was measured by scintillation detection. For detection of VEGF (n=3) and SDF-1α (n=6) supernatant was measured by enzyme linked immuno sorbent assay (ELISA) (ELISA kits and growth factors were from R&D Systems). A BioScaffolder pneumatic dispensing system (SYS+ENG) was used for 3D printing of alginate scaffolds. Models of the scaffold were loaded via CAD/CAM software and translated into a layered model, dispersed by the BioScaffolder [1]. Gelatin microparticles (5 mg/ml) were imbedded into the alginate hydrogel (4% v/v) and printed together with the scaffolds. After printing, scaffolds were dehydrated and embedded in paraffin, then stained with hematoxylin/eosin and Safranin-O. RESULTS Microparticles in the size range of 75-125 µm ∅ were obtained after sieving (Figure 1). In figure 2 the release profiles of the growth factors are shown. Release of BMP-2 from GMPs was gradual and long lasting as we intended. SDF1-α release shows a burst

release during the first day and a more gradual release after that. VEGF-release mainly occurs in a burst within the first eight hours. We successfully printed the GMPs and cells in alginate constructs using the Bioscaffolder. The microparticles were evenly dispersed throughout the hydrogel, their shape unaltered (Figure 3).

DISCUSSION AND CONCLUSION The main advantage of GMPs for growth factor delivery is gradual degradation and growth factor release. Most other growth factor delivery vehicles, for example poly lactic-co-glycolic acid (PLGA) exhibit a larger burst release. The release of growth factors from the gelatin microsphere system is very suitable to use in a 3D scaffold with defined architecture. It enables the release of a growth factor at a certain moment from a certain location, which brings us to a new level of scaffold organization. Regional differences within the scaffold can be defined, which can result in simultaneous MSC recruitment, and bone and vessel formation, each in their own ideal location [1]. In conclusion, combination of 3D fiber deposition with GMPs enables production of scaffolds that show controlled release of growth factors in both time and space. SIGNIFICANCE Improving 3D printing with controlled release of growth factors will result in scaffolds with very precise architecture and properties. This development is an important step towards the production of cell-free constructs to promote recruitment of resident cells as well as bone and vessel formation. ACKNOWLEDGEMENTS This research forms part of the Project P2.04 BONE-IP of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation.

REFERENCES 1. Fedorovich, N.E., et al., Distinct tissue formation by heterogeneous printing of osteo- and endothelial progenitor cells. Tissue Eng Part A, 2011. 17(15-16): p. 2113-21. 2. Ratanavaraporn, J., et al., Synergistic effects of the dual release of stromal cell-derived factor-1 and bone morphogenetic protein-2 from hydrogels on bone regeneration. Biomaterials, 2011. 32(11): p. 2797-811. 3. Kempen, D.H., et al., Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration. Biomaterials, 2009. 30(14): p. 2816-25.

Paper No. 0240 • ORS 2012 Annual Meeting